1. Field of the Invention
This invention relates to methods for detecting specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic, pre-malignant or proliferative disease. Specifically, the invention relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA including non-mutated tumor-associated DNA, and to methods of detecting and monitoring extracellular tumor-associated DNA found in the plasma or serum fraction or non-cellular fraction of blood. Methods may optionally include use of DNA enrichment methods, wherein enrichment-based extraction methods are used prior to amplification and/or detection, or wherein enrichment for the nucleic acid of interest occurs during amplification, in particular through use of a restriction endonuclease. The invention permits the detection of extracellular, tumor-associated nucleic acid in the serum or plasma of humans or other animals having a malignant disease or a pre-malignant or proliferative disease or condition, and is further useful in individuals without a prior diagnosis of neoplastic, pre-malignant or proliferative disease or condition in whom the disease or condition is unrecognized. The invention provides the ability to detect extracellular nucleic acid associated with neoplasia, including but not limited to mutated oncogenes and hypermethylated DNA. The invention thereby provides methods for identification and monitoring of neoplasms and premalignant conditions including but not limited to those of the colon, rectum, pancreas, lung, breast, bladder, ovary, cervix, endometrium, liver, prostate, esophagus, head and neck, and stomach. Methods are thereby further provided for the selection of patients for therapies, monitoring of therapies, and monitoring for tumor recurrence. The invention particularly provides methods for detecting tumor-associated DNA associated with the tyrosine kinase pathway, thereby allowing patient selection for and monitoring of therapies directed toward interfering with the tyrosine kinase pathway and tyrosine kinase receptors, including but not limited to tyrosine kinase inhibitors.
2. Description of the Related Art
Neoplastic disease, including most particularly that collection of diseases known as cancer, is a significant part of morbidity and mortality in adults in the developed world, being surpassed only by cardiovascular disease as the primary cause of adult death. Although improvements in cancer treatment have increased survival times from diagnosis to death, success rates of cancer treatment are more closely related to early detection of neoplastic disease that enable aggressive treatment regimes to be instituted before either primary tumor expansion or metastatic growth can ensue. A particular favorable prognosis is achieved if premalignant tissue can be eradicated prior to progression to cancer.
The art recognizes a wide range of DNA alterations associated with cancer and other neoplastic diseases. Such alterations including mutated DNA, such as point mutations, insertions, deletions, and other mutations of oncogenes and tumor suppression genes, translocations, microsatellite alterations, and non-mutated DNA alterations including hypermethylation, hypomethylation, and excess gene copy number. Oncogenes are normal components of every human and animal cell, responsible for the production of a great number and variety of proteins that control cell proliferation, growth regulation, and cell death.
Acquired mutated oncogenes are markers of malignant or premalignant conditions. It is also known that other, non-oncogenic portions of the genome may be altered in the neoplastic state. Nucleic acid based assays as described in the present invention can detect both oncogenic and non-oncogenic DNA, whether mutated or non-mutated.
One group of tumor-associated DNA of particular interest to which the present invention applies are receptor tyrosine kinase-associated DNA, including but not limited to HER-2/neu DNA, epidermal growth factor receptor (EGFR) DNA, c-Kit DNA, and FMS-like tyrosine kinase 3 (Flt3) DNA.
In particular, nucleic acid amplification methods (for example, the polymerase chain reaction) allow the detection of small numbers of mutant molecules among a background of normal ones. While alternate means of detecting small numbers of tumor cells (such as flow cytometry) have generally been limited to hematological malignancies (Dressler and Bartow, 1989, Semin. Diag. Pathol. 6: 55-82), nucleic acid amplification assays have proven both sensitive and specific in identifying malignant cells and for predicting prognosis following chemotherapy (Fey et al., 1991, Eur. J. Cancer 27: 89-94).
Various nucleic acid amplification strategies for detecting small numbers of mutant molecules in solid tumor tissue have been developed, particularly for the ras oncogene (Chen and Viola, 1991, Anal. Biochem. 195: 51-56; Kahn et al., 1991, Oncogene 6:1079-1083; Pellegata et al., 1992, Anticancer Res. 12:1731-1736; Stork et al., 1991, Oncogene 6: 857-862). For example, one sensitive and specific method identifies mutant ras oncogene DNA on the basis of failure to cleave a restriction site at the crucial 12th codon (Kahn et al., 1991, ibid.). Similar protocols can be applied to detect any mutated region of DNA in a neoplasm, allowing detection of other oncogene DNA or tumor-associated DNA. Since mutated DNA can be detected not only in the primary cancer but in both precursor lesions and metastatic sites (Dix et al., 1995, Diagn. Molec. Pathol. 4: 261-265; Oudejans et al., 1991, Int. J. Cancer 49: 875-879), nucleic acid amplification assays provide a means of detecting and monitoring cancer both early and late in the course of disease.
While direct analysis of neoplastic tissue is frequently difficult or impossible (such as in instances of occult, unrecognized disease), peripheral blood is easily accessible and amenable to nucleic acid-based assays such as those mentioned above. Many studies have used nucleic acid amplification assays to analyze the peripheral blood of patients with cancer in order to detect intracellular DNA extracted from circulating cancer cells in patients, including one study which detected the intracellular ras oncogene from circulating pancreatic cancer cells (Tada et al., 1993, Cancer Res. 53: 2472-4). However, it must be emphasized that these studies attempt to use nucleic acid-based amplification assays to detect extracted intracellular DNA within circulating cancer cells. The assay is performed on the cellular fraction of the blood from patients having cancer using the cell pellet or cells within whole blood, and the serum or plasma fraction is ignored or discarded prior to analysis. Since such an approach requires the presence of metastatic circulating cancer cells (for non-hematologic tumors), it is of limited clinical use in patients with early cancers, and it is not useful in the detection of non-hematologic non-invasive neoplasms or pre-malignant states.
The prior art contains disclosure that mutant oncogene DNA could be detected in peripheral blood plasma or serum of cancer patients (see, for example, Sorenson et al., 1994, Cancer Epidemiology, Biomarkers & Prevention 3: 67-71; Vasioukhin et al., 1994, Br. J. Haematol. 86: 774-9; Vasyukhin et al., in Verna & Shamoo (eds), Biotechnology Today, Ares-Serono Symposia Publications, pp. 141-150). Mutant ras oncogenes have been demonstrated in plasma or serum using polymerase chain reaction. However, these reports have also been generally limited to patients with advanced cancer or known disease.
We have recognized that nucleic acid amplification assays can detect tumor-associated extracellular mutated DNA, including oncogene DNA, in the plasma or serum fraction of blood of humans without cancer or known disease (see U.S. Ser. No. 08/818,058, now U.S. Pat. No. 6,156,504, issued Dec. 5, 2000, incorporated by reference), and that this can be accomplished in a clinically useful manner.